1. Field of the Invention
The present invention pertains to a method of cultivating a microorganism, which is capable of producing teicoplanin, and of producing teicoplanin using a culture broth. The present invention provides a method of economically producing highly pure teicoplanin, in which teicoplanin is produced at a stable, neutral pH range.
2. Description of the Prior Art
Recently, it is regarded as a serious problem that antibiotic-resistant microorganisms are spreading due to abuse of antibiotics. With respect to this, teicoplanin, which is a glycopeptide antibiotic, is considered a last defense line to infectious diseases. Teicoplanin acts against gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), coagulase-negative Staphylococcus, Clostridium and Enterococcus. Teicoplanin is a complex of five kinds of A2 components, having different fatty acid branched chains, and A3, having an aglycone structure in which a sugar moiety of N-acyl-β-D-glucosamine, including the fatty acid branched chain, is removed from a basic structure of A2. As used herein, teicoplanin is designated by the teicoplanin A2 complex acting as an effective component.
Generally, a chemical synthetic process or a bio-synthetic process using a culture of microorganism is adopted to produce antibiotics in commercial quantities. The glycopeptide antibiotics have a complicated chemical structure, in which sugar is bonded to a peptide skeleton. Hence, teicoplanin and the glycopeptide antibiotics commercialized as pharmaceuticals are produced according to the bio-synthetic process, which is a so-called fermentation process. In the bio-synthetic process, various impurities, such as medium components and metabolic products, are produced concomitantly with the antibiotic. Accordingly, a number of separation and purification processes are required to purify the antibiotics, which are critical factors in economically producing a highly pure antibiotic.
Teicoplanin, a glycopeptide antibiotic produced from Actinoplanes teichomyceticus, was first reported in J. Antibiotics 31:170–177 (1978). Efforts have been made to separate teicoplanin from a culture broth of microorganism and purify teicoplanin using various processes so as to produce pharmaceutical grade teicoplanin. According to the above journal and U.S. Pat. No. 4,239,751, a culture broth is divided into a mycelial cake and a filtrate. The mycelial cake is extracted with acetone and the extract is extracted again with butanol at acidic pH. The filtrate, made free from the mycelial mass by filtration, is extracted with butanol at acidic pH. Subsequently, the butanol layers are concentrated by vacuum distillation to form precipitates. The precipitates are mixed with each other, and the mixture is purified with Sephadex LH20 column. The eluate from Sephadex LH20 is further purified with an acidic ion exchange resin, such as IR-120 and Dowex 50, and then, the teicoplanin is precipitated at 4° C. However, the process in U.S. Pat. No. 4,239,751 has disadvantages in that the purification method is very complicated. Furthermore, Sephadex LH 20 is too expensive to apply in a large scale production. Another disadvantage is that the recovery and purity of teicoplanin are poor.
Korean Pat. No. 36780 recites a process of directly extracting teicoplanin from a culture broth, in which a water-miscible organic solvent, such as acetone, n-propanol, and acetonitrile, is added without separating mycelia. Furthermore, Korean Pat. No. 118034 discloses a process of producing teicoplanin by directly adding a strongly acidic cation exchange resin, such as Dow XFS-43278.00 and Diaion SK-102, to a culture broth. However, the processes in Korean Pat. Nos. 367890 and 118034 are disadvantageous in that even though a process of directly extracting teicoplanin from the culture broth is more simplified than in the case of U.S. Pat. No. 4,239,751, it is difficult to employ in a production process because a great amount of organic solvent is inevitably used to extract teicoplanin, which may cause environmental pollution. In addition, it is difficult to produce highly pure teicoplanin by only modifying the extraction process.
Many studies have been carried out to purify teicoplanin by column chromatography using synthetic resins, as suggested by Heydorn et al., J. Biochem. 275:6201–6206 (2000), and as disclosed in European Pat. No. 241,758, Korean Pat. No. 321304, Korean Pat. Laid-Open Publication No. 2003-0017067, and Korean Pat. Laid-Open Publication No. 2003-0034949. Additionally, European Pat. No. 241,758 discloses a process of purifying teicoplanin using a polyamide resin. Further, Korean Pat. No. 184644 discloses an extraction process of teicoplanin from mycelium at alkaline pH, thereby simplifying the complicated extraction process of U.S. Pat. No. 4,239,751. In Korean Pat. No. 184644, after extraction of teicoplanin, the basic culture broth is neutralized, and then purified using the polyamide resin according to a procedure of European Pat. 241,758. However, when the purified teicoplanin was analyzed by HPLC (high performance liquid chromatography), the purity of teicoplanin was not more than 85% and decolorization was poor. Therefore, a further purifying process is required so as to produce highly pure teicoplanin. In order to study a biosynthetic pathway of teicoplanin, Heydorn et al. had separated and purified teicoplanin according to a chromatography process using an ion-exchange resin (Amberlite IRA958) and a hydrophobic adsorption resin (Diaion HP2MGL). However, the method suggested by Heydorn et al. is disadvantageous in that it is inconvenient because acetic acid has to be continuously added to the basic solution passing through the resin to neutralize it and prevent an epimerization. Additionally, when the purity of teicoplanin is analyzed by HPLC after the purified solution is desalted, concentrated, and lyophilized to produce teicoplanin powder, the purity is only about 50% to 60% (w/w). Accordingly, it is undesirable to use the teicoplanin powder as a pharmaceutical ingredient.
Meanwhile, porous adsorption resins have been frequently used to purify glycopeptide antibiotics, including teicoplanin. In detail, Korean Pat. No. 321304 discloses a process of purifying teicoplanin, which includes a hydrophobic interaction chromatography step using a neutral adsorption resin and a lectin-immobilized affinity chromatography step. At this time, the neutral adsorption resin includes XAD 16, HP 20, silica gel, and activated carbon. In this patent, a filtered culture broth is directly purified by the hydrophobic adsorption chromatography using HP-20 and the like, and thus, it is convenient to conduct the process. However, in case that the filtered culture broth extracted from a basic solution is adsorbed into a resin, such as HP 20, according to the process of Korean Pat. No. 321304, a great amount of teicoplanin is lost in an adsorption step, and the purity of teicoplanin eluted by a methanol concentration gradient is very low. Moreover, it is necessary to remove methanol in order to apply the solution to a lectin-immobilized resin. Furthermore, it is not desirable to apply the solution to a lectin-immobilized resin in the large scale production because of the cost of lectin-immobilized resins.
According to Korean Pat. Laid-Open Publication No. 2003–0017067, after teicoplanin of a culture broth is adsorbed into a porous adsorption resin, the porous adsorption resin is washed with diluted hydrochloric acid, and teicoplanin is desorbed from the adsorption resin using a mixed solution of water and acetone. The eluting solution containing teicoplanin is concentrated by vacuum distillation, treated with an activated carbon, and subjected to a precipitation process, and thereby teicoplanin is purified. However, the process in Korean Pat. Laid-Open Publication No. 2003-0017067 is disadvantageous in that the stability and activity of teicoplanin are decreased because the pH of the liquid that is processed is continuously changed to acid or basic. Other disadvantages are that the life-time and exchange cycle of the resin are shortened, and recovery yield and purity of teicoplanin are poor because of moieties irreversibly adsorbed into the resin. Meanwhile, Korean Pat. Laid-Open Publication No. 2003-0034949 discloses a method of producing teicoplanin, which includes roughly purifying teicoplanin from a culture broth through a two-stage process using porous adsorption resins, and precipitating teicoplanin at low temperatures and slightly acidic pH. However, this method has disadvantages in that the use of a large amount of organic solvent, such as n-propanol, isopropanol, and methanol, contributes to pollution, and that precipitation at low temperature and slightly acidic pH reduces the solubility and activity of teicoplanin.
Furthermore, it is difficult to purify teicoplanin of 95% or higher purity through only a purifying process using porous adsorption resins. Accordingly, many studies have been carried out with reverse phase resins to separate and purify teicoplanin from the culture broth. For example, references can be made to a process suggested by Riva et al., Chromatographia 24:295–301 (1987), Korean Pat. No. 40453, and Korean Pat. Laid-Open Publication Nos. 2003-0092504 and 10-2004-0008745. Riva et al. proposed a process of purifying teicoplanin using a Lichrosorb RP-18 column. Korean Pat. No. 40453 discloses a process of separating each single component of teicoplanin A2 complex using a silanized silica gel column. At this time, in the case of using the reverse phase resin, it is possible to produce more pure teicoplanin than in the case of a separation process using a combination of an extraction, an ion-exchange resin, and a porous adsorption resin. However, Korean Pat. No. 40453 is problematic in economic efficiency because the reverse phase resin and high pressure chromatography system are both costly. Further, acetonitrile, which is toxic to the human nervous system, is used in eluting teicoplanin from the reverse phase resin in Korean Pat. No. 40453. Furthermore, Korean Pat. Laid-Open Publication No. 2003-0092504 proposes a method of purifying teicoplanin, in which a mycelium-free culture broth directly passes through a reverse phase resin, such as YMC-gel ODS-A, or in which a roughly purified liquid, pre-treated with a cation-exchange resin, an anion-exchange resin, or a adsorption resin, passes through YMC-gel ODS-A. However, this method is disadvantageous in that acetonitrile is used for the elution, and thus, it is difficult to control the residual amounts of acetonitrile. Another disadvantage is that production costs are inevitably increased because the reverse phase resin must frequently be replaced with a new one. Korean Pat. Laid-Open Publication No. 10-2004-0008745 recites a process of purifying teicoplanin from a culture broth of a microorganism capable of producing teicoplanin, which includes a primary purifying step using a synthetic adsorbent, a secondary purifying step using a cation-exchange resin, a catalytic resin, or a chelate resin, a tertiary purifying step using a reverse phase resin, and a final lyophilization step. However, the process in Korean Pat. Laid-Open Publication No. 10-2004-0008745 is disadvantageous in that even though highly pure teicoplanin is produced, the process is very complicated because the process includes a number of steps, and recovery yield of teicoplanin is very low. Moreover, the process in Korean Pat. Laid-Open Publication No. 10-2004-0008745 has the same disadvantages, regarding the use of the reverse phase resin, as the process in Korean Pat. Laid-Open Publication No.2003-0092504.
Accordingly, conventional technologies of purifying teicoplanin from the culture broth are problematic in that highly purified teicoplanin is not readily produced, stability of teicoplanin is not maintained, the organic solvents toxic to humans are used during the purification, recovery yield is relatively low, and production costs are relatively high. Hence, there remains a need to develop an improved process of purifying teicoplanin.